US10519414B2 - Apparatus and method for continuous cell culture - Google Patents

Apparatus and method for continuous cell culture Download PDF

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Publication number
US10519414B2
US10519414B2 US15/027,751 US201415027751A US10519414B2 US 10519414 B2 US10519414 B2 US 10519414B2 US 201415027751 A US201415027751 A US 201415027751A US 10519414 B2 US10519414 B2 US 10519414B2
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cells
culture
compartment
culture vessel
scraper
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US20160237392A1 (en
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Hee Young Lee
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MEDIKAN Inc
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MEDIKAN Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/16—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature by recirculation of culture medium at controlled temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/24—Recirculation of gas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26—Conditioning fluids entering or exiting the reaction vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • a separate sealing unit needs to be provided when the cultured cells are moved to the outside of a laboratory, but there is no successful case where sealing is perfect, and vessels are mostly transported in order to move the cells.
  • the enzyme dissolves the connection surface with the adhesion surface to destabilize adhesion of the cells, and entails various problems such as a long enzyme treatment time, changes in the properties of the cells and the death of cells owing to toxicity of the enzyme.
  • an object of the present invention is to provide a device for continuously culturing cells and a culturing method using the same, in which the cells and a culture medium are selectively and repeatedly injected, cultured, detached, and obtained while a sealed state of a culture vessel is maintained, and accordingly, the cells are obtained while some of the cultured cells are allowed to remain during an obtaining process, thereby repeatedly culturing the cells remaining in the culture vessel, preventing the cultured cells from being contaminated, and attaining uniformity of the cultured cells.
  • the present invention provides a device for continuously culturing cells, including a culture vessel having a compartment that is selectively determined to be sealed.
  • a culture medium is injected into the compartment and inoculated with the cells and the cells are detached and obtained while the compartment is sealed, and the cells are cultured while the compartment is open.
  • negative pressure is generated in a culture environment unit including the culture vessel stored therein to change the size of the compartment of the culture vessel to thus supply the gas required to culture the cells into the compartment in the culture environment unit through the circulation filter.
  • the scraper is rotated or moved while in contact with the internal surface of the compartment using magnetic force to scrape the cells.
  • a culture groove is formed on the scraper to be filled with a culture medium and culture the cell.
  • the scraper is made of a material selected from polyethylene (PE), polypropylene (PP), polyamide (PA), polyacetal (POM), polyvinyl chloride (PVC), polyester (PET), polymethylpentene (PMP), an ionomer (IO), ethylene vinyl alcohol (EVOH), polystyrene (PS), a methacrylic resin (PMMA), polycarbonate (PC), polyvinyl acetate (PVAc), polyvinyl alcohol (PVA), a phenol resin (PF), a urea resin (UF), a melamine resin (MF), an epoxy resin (EP), polyurethane (PUR), an unsaturated polyester resin (UP), and a metal.
  • PE polyethylene
  • PP polypropylene
  • PA polyamide
  • POM polyacetal
  • PVC polyvinyl chloride
  • PET polyester
  • PMP polymethylpentene
  • IO ethylene vinyl alcohol
  • EVOH ethylene vinyl alcohol
  • PS polystyrene
  • the scraper and the moving member are linked using magnetic force.
  • the propagating of the cells in the culture medium includes inoculating the culture medium with the cells and then storing a culture vessel in a culture environment unit while the culture environment unit has a culture temperature of 0 to 42° C. and a gas, required to culture the cells, is supplied to the culture vessel to provide an optimum culture environment, thus culturing the cells.
  • a sealed culture vessel is selectively opened and closed to an outside, and the culture vessel is drawn from a culture environment unit and conveyed after the compartment of the sealed culture vessel is sealed to the outside.
  • the cells and a culture medium are selectively and repeatedly injected, cultured, detached, and obtained while a seal of a culture vessel is maintained, and accordingly, the cells are obtained while some of the cultured cells are allowed to remain during an obtaining process, thereby repeatedly culturing the cells remaining in the culture vessel, preventing the cultured cells from being contaminated, and attaining uniformity of the cultured cells.
  • FIG. 10 is a perspective view of a scraper of a device for continuously culturing cells according to another embodiment of the present invention.
  • FIG. 13 is a perspective view of a device for continuously culturing cells according to another embodiment of the present invention.
  • Culture device 11 Cell 12: Culture medium 100: Culture vessel 101: Space 110: Sealing-type passageway 120: Circulation filter 121: Conduit 122: Valve 123: Filter 124: Clip 130: Scraper 131: Blade 132: Culture groove 133: Central axis 134: Metal body 140: Moving member 141: Magnetic substance 200: Culture environment unit
  • FIG. 1 is a perspective view of a device for continuously culturing cells according to the present invention
  • FIG. 2 is a longitudinal sectional view of the device for continuously culturing the cells according to the present invention
  • FIG. 3 is a transverse sectional view of the device for continuously culturing the cells according to the present invention
  • FIGS. 4 to 9 are flowcharts showing culturing of the cells using the device for continuously culturing the cells according to the present invention
  • FIG. 10 is a perspective view of a scraper of a device for continuously culturing cells according to another embodiment of the present invention
  • FIG. 11 is a perspective view of a scraper of a device for continuously culturing cells according to various embodiments of the present invention
  • FIG. 12 is a perspective view of a culture environment unit including the device for continuously culturing cells stored therein according to the present invention
  • FIG. 13 is a perspective view of a device for continuously culturing cells according to another embodiment of the present invention.
  • the gas is moved out of or into the compartment 101 in the culture vessel 100 through the circulation filter 120 .
  • the culture vessel 100 When the cells 11 are detached using the rotation of the scraper 130 , the culture vessel 100 has a cylinder shape, and the scraper 130 is rotated around the central axis 133 along the internal circumference of the compartment 101 to detach the cells 11 .
  • a moving member 140 which is separately provided, is moved so as to come close to the external lower surface of the culture vessel 100 , the scraper 130 and the moving member 140 are linked using magnetic force, and the scraper 130 is rubbed in the compartment 101 according to the movement of the moving member 140 so as to detach the cells 11 by scraping.
  • the scraper 130 and the moving member 140 may include a metal body 134 or a magnetic substance 141 so as to be integrally linked with each other using magnetic force.
  • the scraper 130 having the aforementioned constitution has a culture groove 132 in the upper side thereof to fill the culture groove with the culture medium 12 and culture the cells 11 in the culture groove.
  • the culture medium 12 is inoculated with the cells 11 and then the culture vessel is stored in the culture environment unit 200 to culture the cells.
  • the culture environment unit 200 has a culture temperature of 0 to 42° C., and the gas required to culture the cells 11 is supplied to the culture vessel.
  • pressure from the clip is removed from the circulation filter so as to open the conduit.
  • the scraper 130 is rotatably combined with the compartment 101 through the central axis 133 , and the central axis 133 is exposed to be interlocked with the scraper 130 in the compartment 101 and rotated to thus detach the cells 11 from the bottom surface of the compartment 101 by scraping.
  • the scraper 130 is moved using potential energy to detach the cells 11 from the bottom surface of the compartment 101 by scraping.
  • the needle of the syringe is stuck into one of the sealing-type passageways 110 other than the sealing-type passageway 110 into which the culture medium is injected, so as to draw the cultured cells 11 through the needle into the syringe using the negative pressure of the syringe, thereby obtaining the cells.
  • some of the detached cells 11 are allowed to remain during the obtaining of the cells 11 , so that the remaining cells 11 are repeatedly propagated.
  • the injecting of the culture medium 12 , the inoculating of the culture medium 12 with the cells 11 , the culturing of the cells 11 , the detaching of the cells 11 , and the obtaining of the cells 11 are performed while the sealed culture vessel is always sealed so as to stably and repeatedly culture the cells 11 after the obtaining of the cells 11 .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
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  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Clinical Laboratory Science (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Thermal Sciences (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Water Supply & Treatment (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US15/027,751 2013-10-16 2014-10-16 Apparatus and method for continuous cell culture Active 2035-11-29 US10519414B2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
KR10-2013-0123459 2013-10-16
KR20130123459 2013-10-16
KR1020140139677A KR101731590B1 (ko) 2013-10-16 2014-10-16 연속으로 세포를 배양하는 장치 및 방법
PCT/KR2014/009714 WO2015056986A1 (ko) 2013-10-16 2014-10-16 연속으로 세포를 배양하는 장치 및 방법
KR10-2014-0139677 2014-10-16

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US20160237392A1 US20160237392A1 (en) 2016-08-18
US10519414B2 true US10519414B2 (en) 2019-12-31

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US (1) US10519414B2 (ja)
EP (1) EP3059301A4 (ja)
JP (1) JP6702859B2 (ja)
KR (1) KR101731590B1 (ja)
CN (1) CN105658783A (ja)
CA (1) CA2924221A1 (ja)

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KR102588616B1 (ko) * 2016-04-06 2023-10-16 메디칸(주) 무전원 정온 세포 이송 장치
WO2018195014A1 (en) * 2017-04-19 2018-10-25 Neubiser Richard Bioreactor for biological material
KR101937039B1 (ko) 2017-07-18 2019-01-09 포항공과대학교 산학협력단 체외 세포 배양 블록
US11643667B2 (en) 2017-08-28 2023-05-09 Cellino Biotech, Inc. Microfluidic laser-activated intracellular delivery systems and methods
CN107445714A (zh) * 2017-09-18 2017-12-08 何玉莲 一种园林栽培用生态修复培养基及其制备方法
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JP2019062855A (ja) * 2017-10-04 2019-04-25 ヨン イ，ヒョ 無電源定温細胞移送装置
CN108485921A (zh) * 2018-07-05 2018-09-04 广州市天河诺亚生物工程有限公司 细胞刮刀及细胞刮取组件
CN110184300A (zh) * 2019-05-21 2019-08-30 安徽环球基因科技有限公司 一种利用稳定细胞株生产膜蛋白的方法
CN110229752B (zh) * 2019-06-24 2024-03-19 上海长海医院 一种微负压细胞培养装置
CN112457988B (zh) * 2020-11-19 2022-08-26 江西迈柯菲生物医药科技有限公司 一种立体式大规模贴壁细胞培养装置
WO2022192157A1 (en) 2021-03-07 2022-09-15 Cellino Biotech, Inc. Platforms and systems for automated cell culture
US11931737B2 (en) 2021-09-02 2024-03-19 Cellino Biotech, Inc. Platforms and systems for automated cell culture
CN113894904A (zh) * 2021-09-23 2022-01-07 大亚人造板集团有限公司 一种薄型刨花板的生产方法

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KR101731590B1 (ko) 2017-05-02
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CN105658783A (zh) 2016-06-08
JP6702859B2 (ja) 2020-06-03

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